2011 (SBTE) 25th Annual Meeting Proceedings - International ...

Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011.
A209 CLONING, TRANSGENESIS AND STEM CELLS
IN VITRO CULTURE AND OSTEOGENIC DIFFERENTIATION TION OF CRYOPRESER
OPRESERVED CANINE ADIPOSE-DERIVED STEM CELL
Isadora Arruda, Camila Chavier Macedo, Amanda Jeronimo Listoni, Midyan Daroz Guastali, Margarita Pardo, Bruna de Vita, Leandro Maia & Fernanda da
Cruz Landim Alvarenga
UNESP - FMVZ, BOTUCATU, SP, BRAZIL.
The utilization of canine adipose-derived stem cell (adMSC) has raised great interest in the scientific community due to its amazing
regenerative potential, especially for the treatment of osteogenic and tendinous lesions. The adMSC had the advantage of been easily collected,
appearing in abundance in the tissue of origin. However, the protocols of isolation, culture and cryopreservation are not well established in all
domestic animals. The objective of the present study was to verify the potential of in vitro culture and osteogenic differentiation of cryopreserved
adMSC visceral fat obtained from dogs. Fragments from visceral fat were obtained from the mesentery during cesarean sections and the cells
were obtained after enzymatic digestion with 0.4% colagenase (Gibco ® , NY/USA). Isolation and expansion were performed in DMEM-F12
with fetal calf serum (FCS) supplemented with antibiotics and antimycotics (Gibco ® , NY/USA) at 37°C with 5% CO2. The adMSC in 1st and
3rd passage were cryopreservated in media with 10% DMSO (Sigma ® , St. Louis/USA) at -80°C. After 8 days, the samples were thaw in water
bath at 37°C for 2 min, centrifuged, diluted and seeded in new bottles. The differentiation of the samples in P3 was performed as soon as the
culture reached 80% confluence but the cells in P1 were kept in culture until the 3rd passage and then differentiated. For differentiation, the media
was replaced by the Mesenchymal Stem Cell Osteogenesis Kit (Chemicon ® , MA/USA) and StemPro Mesenchymal Osteogenesis Kit (Gibco ® ,
NY, USA). The media was changed every two days and the confirmation of the osteogenic differentiation was performed on day 9 by staining
the samples with Alizarin Red (Sigma ® , St. Louis/USA), who stains the extracellular deposit of calcium in red. All samples were positive for
calcium deposit. So, it was possible to conclude that the culture and osteogenic differentiation of adMSC obtained from visceral fat from dogs
was efficient even after cryopreservation at -80°C for 8 days. Moreover, the both commercial kits used in this experiment were efficient in
inducing osteogenic differentiation in the dog cells utilized.
Keywords: stem cell, cryopreservation, canine.
A210 CLONING, TRANSGENESIS AND STEM CELLS
EFFECT OF FIBROBL
OBLAST CELL PASSA
ASSAGE ON PRODUCTION OF GYR EMBRYOS BY NUCLEAR TRANSFER
TECHNIQUE
C arolina Cap
apobiango R. Quin
uintão
1 , M ichele Munk Per
ereir
eira 1 , Na tana Cha
hav es Rab
abelo
1 , Jorje R. Toledo Alonso
2 , Lilian Tam
amy Iguma 1 João
Henrique Moreira Viana 1 & Luiz Sérgio Almeida Camargo 1
1
EMBRAPA GADO DE LEITE, JUIZ DE FORA, MG, BRAZIL. 2 UNIVERSIDAD, DE CONCEPCIÓN, CHILE.
The success of the somatic cell nuclear transfer (SCNT) depends, among other factors, on donor cell competence to undergo nuclear
reprogramming and generate clone embryos. Such competence can be influenced by the cell passage (Kubota et al. 2000; PNAS 97:990-995).
This study aimed to evaluate the effect of cell passage of fibroblasts from Gyr cow to produce clone embryos. Recipient oocytes were obtained
from abattoir ovaries and matured in TCM 199 (Invitrogen, California, USA) supplemented with 10% estrous cow serum (ECS) under 5% CO2
and 95% humidity at 37°C in air. After maturation, a portion of oocytes was denuded and enucleated for SCNT and the remainder was used
for in vitro fertilization (IVF). Bovine fibroblasts collected from Gyr cows were cultured for several passages in DMEM supplemented with
10% fetal calf serum (FCS) and incubated at 37°C, 5% CO2 and 95% humidity. Three experimental groups were established as follow: G1-
enucleated oocytes fused with bovine fibroblasts at 3rd - 4th cell passagel; G2- enucleated oocytes fused with bovine fibroblasts at 6th - 8th cell
passage; and G3- oocytes fertilized in vitro with 2 x 106 sperm/mL for 21h. For G1 and G2, the fibroblasts were cultured for 48h in total
confluence before being trypsinized and used for SCNT. Fibroblasts from G1 and G2 were fused with enucleated oocytes using two pulses of
2.4 kV/cm for 30 msec and chemically activated with ionomycin (Sigma, St. Louis, USA) followed by DMAP (Sigma). Zygotes from all groups
were cultured in CR2aa medium with 2.5% FCS under 5% CO2, 5% O2 and 90% N2 at 38.5ºC. Cleavage was evaluated at 72h and blastocyst
at 168h post-activation (G1 and G2) or post-fertilization (G3) and the data were analyzed by chi-square. No significant differences (P > 0.05)
in cleavage between G1 (84.74%) and G2 (84.31%), but the blastocyst rate was higher (P < 0.05) for G2 (39.2 %) than for G1 (26.2%). The
cleavage (66.67%) and blastocyst (13.70%) rates were lower for G3 than for other groups (P < 0.05) in. In conclusion, bovine fibroblasts at
earlier passages (3rd-4th passage) of cell culture are less effective in producing Gyr embryos by nuclear transfer when compared to later passages
(6th-8th passage). However, it is noteworthy that embryos produced with deriving cells of a long period of culture may have affected their quality
(Merighe et al. 2010). [Financial support: CNPq – FAPEMIG and Innovation Network Project on Animal Reproduction].
Keywords: clone, somatic cell, cell passage.
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